Transcription

1 IRACST Engineering Science and Technology: An International Journal (ESTIJ), ISSN: , Histopathological Examination of araphenylen Diamine Toxicity in Female Rats Liver Aisha D. Alalwani Assistant rofessor Histology and Cytology department Science Faculty for Girls, King Abdulaziz University, Jeddah, Saudi Arabia. Abstract araphenylendiamine (D), a widely used in a variety of industrial products and in almost all hair dye formulation has been tested for its hepatotoxicity after 6 months topical application in two different dosages (0.5 and 1mg/kg/day/week) in female Wistar rats. All serum biomarker of liver toxicity aspartate transaminase, alanine transaminase and alkaline phosphatase shows a dose dependent increase in the D treated group of animals, as well as mean body weights and the relative liver weights were affected. The histopathological findings in group III included congestion of central and portal veins, with cellular infiltration. Disruption of blood sinusoids, necrotic of kupffer cells, as well as proliferation of bile ducts, focal necrosis of hepatic cells with deformed nuclei which appeared in pyknosis and karyolysis stages. Vacuolar cytoplasmic degeneration led to focal lysis of liver parenchyma showed in group IV. Electron microscopic changes in the group III confirmed low electron density of hepatocytes, degranulation of rough ER cisterns, replication of smooth ER, proliferation of bile canaliculi. In contrast, D caused various hepatic changes in group IV as nucleus polymorphism with irregular envelope. Slipper shape with cristolysis mitochondria. Necrosis of kupffer and endothelial cells nuclei with spire the membrane. Increased lysosomal numbers. Key words: araphenylendiamine, Hepatotoxicity, Hepatic enzymes, Ultra Structure. I. INTRODUCTION arahenylendiamine (D) is a common ingredient in most of the hair dye preparations. It accelerates the dyeing process and may produce local as well as systemic toxic effects when applied topically and ingested[1]. D is an organic compound. derivative from aniline aromatic amine, is a colourless solid when pure. It is used primarily as a fur and hair dye and as a chemical intermediate in the production of numerous substances, including dyes and polymers [2]. D is commonly used in its raw form for cosmetic purposes in Africa, iddle East and Indian subcontinent while it is rarely used in the west [3,4]. In Sudan, D is mixed with henna, leaves of Lawsonia Alba, which is a non toxic herb used to decorate the hands and feet in special social events, such as wedding ceremonies [4], and over a 10year period ( ), 3159 patients were reported to suffer from D poisoning; among these were 568 (18%) children below the age of 14 years [5,6,7]. A study from orocco described 374 cases of D poisoning in adults and children over a 10year period [5]. A report from Tunisia showed similar results[8], and a report from Saudi Arabia documented a suicide attempt with D in a 14year old female [9,10,11]. Similarly in India, popular hair dyes contain D, among other ingredients[5,12]. Some product sold as henna also contains D articularly black henna[13]. Short exposure to high level of D may cause severe dermatitis, eye irritation and tearing, Asthma, renal failure, vertigo tremers, convulsions and coma. Ingestion of D produces rapid developments of edema of face, neck, pharynx tongue and larynx with respiratory distress which often needs tracheostomy[1]. In the later stages Rhabdomyolysis and acute tubular necrosis with acute renal failure and hepatic failure develops, bleeding tendency (bleeding from gums), subconjunctival hemorrhage and bleeding from mucus membrane also occur [13]. athological changes in mitochondria as swollen or atrophy,cavitations within the mitochondria, considered that an important indicator of cellular damage leading to loss of functional efficiency, added that the mitochondria destruction may be due to disturbance of calcium ions Ca+2 into cells, or block the AT synthesis and thus block the phosphorous oxidation in mitochondria, or may be due to loss of mitochondria membrane permeability or change the cellular H [14]. Few studies were identified that investigated the dermal absorption potential of D in humans and animals. Under exposure conditions that mimicked the intendeduse conditions for such hair dyes [15]. Under the intendeduse conditions, dermal absorption of 0.54% to 2.7% in volunteers [16,17,18,19] and 2.7% in monkeys [16,18,20] has been reported. Dermal absorption of 2.7% has been noted in human cadaver skin [6,10]. The degree of dermal absorption was reported as 0.93% [18] in excised pig skin [19] reported similar dermal absorption values, of 2.44% and 3.39% in vitro, in human and pig skin, respectively. In addition tattoos are very popular in Arabian Gulf, especially in Saudi Arabia. Since, tattoo industry is not regulated, people are still getting black henna tattoos and exposing themselves to serious toxicity problems [14]. A very few report are available that pointed towards the hepatotoxicity by hair dye ingredients [21], and chronic dermal exposure and subsequent histopathological alterations of liver tissue by D substance. Therefore the present study was conducted to analyze dose dependent hepatic effect of repeated topical application of araphenylen Diamine on hepatocytes structure and liver function of female rats. 296

2 II. A. Chemical phenylendiamine (D) light purple powder, purchased from commercial store in Jeddah of Saudi Arabia. CAS No : , Batch: 99E483. olecular weight (W): 108 and 98% purity was. C6H8N2 (free base), C6H8N2. 2HCl (dihydrochloride),c6h8n2. H2SO4 (sulfate) (OECD,2010a ; Hummdi,2012). Structural Formula IRACST Engineering Science and Technology: An International Journal (ESTIJ), ISSN: , ATERIALS AND ETHODS rotator microtome. The deparaffinized sections were stained routinely with hematoxylin and eosin [27]. hotomicrographs of the desired sections were made by light microscope for further observations. E. Electron icroscopy Samples of liver were fixed in 2.5% glutaraldehyde and 0.25 sodium cacodylate, postfixed in 1% osmium tetroxide, and embedded in Spurr's epoxy. Ultrathin sections were picked up on nickel grids, stained with uranyl acetate/lead citrate [28], and examined in a hilips TE 100 microscope. F. Statistical Analysis B. Animals and their treatment The study was conducted according to OECD [22,23] on the design and conduct of chronic toxicity and the CE [23] recommendations on rodents housing, Wistar rats (Rattus norvegicus albinus) 35 weeks old female rats, (n=80), with an average body weight of about ±7.10 g were obtained from the animal house of the King Fahd Center for edical Research, King Abdul Aziz University in Jeddah of Saudi Arabia. The Animals housed under a 12 h light/12 h dark cycle in stainless steel cages and allotted randomly to four groups(n=20). Group I and II was the control groups, while groups III and IV were topical application of or ml (0.5 and 1 mg/kg/bw) respectively of D dissolved in double (degassed purified water) in a shaved of approximately 10% of the total body surface area of inter scapular skin for 30 minutes per day, 7 days per week, for a period of 6 months [23,24,25]. Since, the control groups received degassed purified water painted on their dorsal side as in other D treated animals. The rats were weekly weighed and observed daily for signs of toxicity and for mortality. C. Biochemical assays At termination of the study, the blood samples were collected 24 hours after the experiment and analyzed for aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (AL) by using the commercial kits according to procedures. [26] procedures. D. Histological study After bleeding, the rats were sacrificed by cervical dislocation and the abdominal cavity was opened up to expose the liver which were quickly dissected out, weighted and fixed in 10% formal saline for routine histological techniques. The tissues were dehydrated in an ascending grade of alcohol (ethanol), cleared in xylene and embedded in paraffin wax. Serial sections of 2 microns thick were obtained using a ean ± standard error values were calculated for each group. Differences in the experimental data were analyzed using Student s ttest. values <0.05were considered to be significant. All statistical calculations were performed using SSS 18.0 software for Windows. III. RESULT A. Clinical signs and mortality ortality and clinical signs of each groups were significantly associated with the concentration of D administered table 1. Table 1:Clinical symptoms and the mortality recorded in groups 3 and 4 of D treatment rats after 24hr 6 months. Clinical symptoms Swelling face and neck Dark discoloration of urine No. of Experimental animals =20 B. Body and liver weights No. of Experimental animals =20 N0.of N0.of individual individual 17 85% 19 95% Significant differences were observed in the terminal body weight gain among the treated and control group of animals. Both absolute and relative weight exhibits significant dose response increase after D treatment compared to controls table 2. % 17 85% 18 90% Ataxia 13 65% 15 75% Skin 6 30% 10 50% changes(der matitis) ortality 2 10% 7 35% % 297

6 lesions in both rats treated groups in this study. while, AST and ALT associated with liver parenchyma cells so they are raised in acute liver damage, following the level decrease gradually in chronic diseases or toxic substance [31,32],and supported the hepatocellular injury damage bile canalicular microvilli with deformation of surrounding desmosomes in recent work. The mechanism of hepatocytes toxicity may results either directly from the disruption of intracellular function or membrane integrity or from damages affecting endothelial or bile duct cells or indirectly from immune mediated membrane damage [33]. As were appeared in the present study the prolonged exposure to the toxin turned the degenerative changes into necrotic damage and tissue lysis. This result is confirmed by [34] who suggested that the cellular degeneration might be attributed to liberation of acid hydrolyses released from the destructed lysosomes to facilitate the process of autolysis. However, in the current work, pyknosis and karyolysis of cell nuclei may indicate the loss of functional efficiency of the cells. Similar results have been demonstrated by [35].It was evident that the degenerative changes appeared earlier in the cytoplasm than in the nuclei of hepatocytes. This result is consistent with the findings of [36] who stated that the nuclear damage is a sequence of cytoplasmic damage. In contrast, the cytoplasmic vacuolation were showed in group IV treated rats. Similarly[3] and [14] described sever tubular necrosis in kidney D poisoning as vacuolar degeneration, nuclear pyknosis and cytoplasmic vacuolations brush border damage. reviously [36] study described the vacuolation of hepatocytes as ballooning degeneration and interpreted it as a kind of cellular defensive mechanism against injurious substances. [37] added these vacuoles are responsible for collecting the injurious elements and preventing them from interfering with the biological activities of these cells. However, the present results are consistent with the [38] who indicated that the cell swollen may be due to inhibition of the production of energy as a result of the damage mitochondrial crista and increase the permeability of the cell plasma membrane[39]. Additionally, in early stages of hepatotoxicity proliferation of the smooth endoplasmic reticulum (SER) can be seen to face the adverse effects associated with an increase of detoxification enzymes, and with ongoing cellular toxicity the SER comes to occupy most of the cell, both by proliferation,and dilatation of its cisternae with vesiculated [37]. oreover, the proliferation of RER is the compensate activity for the deterioration of the cellular protein when cells are exposed to various toxins [40]. Acute and chronic hepatitis is pathologically characterized by a prominent infiltration of lymphocytes into the liver [41] and this histological feature is predominantly found in the liver of D treated rats. The main encountered inflammatory cells in the recent work was macrophages and lymphocytes which predominant in intoxication, viral and protozoal diseases, cause that the lymphocytes produce antitoxins and accelerate cell healing, whereas, the macrophages IRACST Engineering Science and Technology: An International Journal (ESTIJ), ISSN: , destroyed the causes of damage and injured tissues [42]. Furthermore, [37] reported that the inflammation is accompanied by lethal damage to endothelial cells, loss of tone leads to marked expansion of the blood vessels and packing of the lumen with erythrocytes. On the contrary, the current work revealed Kupffer and endothelial cells necrosis, theses finding consistent with [43]study were attributed. [44] added the D induces free radicals subsequently results in lipid peroxidation [43], which led to liver cells necrosis, and renal tissue necrosis [14,42]. However, the proliferation of bile ducts in this work is in agreement with [45] who stated that the cells lining the bile ducts are stem cells that activated and prolifered with necrosis or lysis of liver cells. V. CONCLUSION The doses (0,5 and 1 mg kg) selected in the present work for 6 months revealed liver toxicity and ultrastructure alterations resulting in impairment of liver function. Further experimental information is necessary to confirm the role and mechanism of D mediated hepatotoxicity. REFRANCES 1. Neelima S, Jatav O, Gupta RK, Tailor T (2008). yocardial Damage in Hair Dye oisoning An Uncommon resentation. JAI. 56: HSDB (Hazardous Substance Data Bank) (2009). henylene diaminedialine. [ Accessed Chugh KS, alik GH, Singhal C (1982). Acute renal failure following paraphenylene diamine [Hair dye] poisoning: report of two cases. J. ed.13(12): Sir Hashim, Hamza YO, Yahia B, Khogali F, Sulieman GI (1992). oisoning from henna dye and paraphenylenediamine mixtures in children in Kartoum. Ann. Trop. aediatr.12(1): Filali A, Semlali I, Ottaviano V, Fumari C, Corradini D, Soulaymani R (2006). A retrospective study of acute systemic poisoning of paraphenylenediamine (occidental takawt) in orocco. Afr. J. Trad. 3(1): Hamdouk I, Abdelraheem B, Taha AA, Benghanem, De Broe (2008). araphenylene diamine hair dye poisoning. In: De Broe E, orter GA, Bennett W, Deray G, editors. Clinical nephrotoxins: renal injury from drugs and chemicals. 3 rd ed. NewYork: Springer. p Abdelraheem B, ElTigani A, Hassan EG, Ali A, ohamed IA, Nazik AE (2009). Acute renal failure owing to paraphenylene diamine hair dye poisoning in Sudanese children. Ann. Trop. aediatr.29(3): Kallel H, Chelly H, Dammak H, Bahloul, Ksibi H, Hamida CB, Chaari A, Rekik N, De Broe, E, Bouaziz (2005). Clinical manifestations of systemic paraphenylene diamine intoxication. J Nephrol. 18(3): Ashraf W, Dawling S, Farrow LJ (1994). Systemic paraphenylene diamine (D) poisoning: a case report and review. Hum. Exp. Toxicol.13(3): Dresseler WE, Appelqv IS (2006). lasma /blood pharmacokinetics and metabolism after dermal exposure to paraaminophenol or paraphenylenediamine. Food Chem. Toxicol. 44:

Lecture 5: Cellular Biology I. Cell Theory Concepts: 1. Cells are the functional and structural units of living organisms 2. The activity of an organism is dependent on both the individual and collective

Overview of the Cellular Basis of Life Cells and Tissues Cells: Carry out all chemical activities needed to sustain life Cells are the building blocks of all living things Tissues Cells vary in length,

2 Yara shwabkeh Osama Alkhader Heba Kalbouneh CELL OVERVIEW -Note ; the important thing is to know how the organelles appear under the microscope - the stains we usually use in Histology are composed of

Many of the histologic images and the tables are from MacSween s Pathology of the Liver (5 th Edition). Other images were used from an online source called PathPedia.com. A few images from other sources

Part I. Clinical Applications Name: The Cell Study Guide, Chapter 2 Lab Time: 1 1. Johnny lacerated his arm and rushed home to Mom so she could fix it. His mother poured hydrogen peroxide over the area,

Slide 154: Pancreas, H&E the pancreas, located adjacent to the duodenum, is a mixed exocrine and endocrine gland; it is usually readily identifiable by the presence of the interspersed endocrine pancreatic

3- Cell Structure and Function How do things move in and out of cells? A Quick Review Taft College Human Physiology How do things move in and out of cells? Things may move through cell membranes by Passive

Question 1: Who discovered cells and how? Cells were discovered in 1665 by an English Botanist, Robert Hooke. He used a primitive microscope to observe cells in a cork slice. Question 2: Why is the cell

Cell Structure and Function Biology 12 Unit 1 Cell Structure and Function Inquiry into Life pages 45 59 and 68-69 Assignments for this Unit Pick up the notes/worksheet for this unit and the project There

1 Class IX Chapter 5 The Fundamental Unit of Life Science Question 1: Who discovered cells and how? Cells were discovered in 1665 by an English Botanist, Robert Hooke. He used a primitive microscope to

Lymphoid Organs Dr. Sami Zaqout Cells of the Immune System Lymphocytes Plasma cells Mast cells Neutrophils Eosinophils Cells of the mononuclear phagocyte system Distribution of cells of the immune system

Salicylate (Aspirin) Ingestion California Poison Control 1-800-876-4766 Background 1. The prevalence of aspirin-containing analgesic products makes these agents, found in virtually every household, common

General and Systemic Histopathology C601 and C602 Section 8 As we will see in this unit, the liver is subject to many types of injury. Additionally, many systemic diseases have a liver component and sometimes

Agents that inhibit the BSEP and mitochondrial function what do we know? Prepared by Michael D. Aleo, Ph.D. Groton, Investigative Toxicology Declarations Nonclinical studies All procedures performed on

Ch. 2 Cell Structure and Func.on BIOL 100 Cells Fundamental units of life Cell theory All living things are composed of one or more cells. The cell is the most basic unit of life. All cells come from pre-existing

Cell Theory 4 basic concepts of cell theory are: Cells are the units of structure (building blocks) of all animals and plants. Cells are the smallest unit of function in all animals and plants. Cells originate

PULMONARY FAT EMBOLISM FOLLOWING INFUSIONS VIA THE BONE MARROW1 IJDO J. WILE, M.D., AND IRA LEO SCHAMBERG, M.D. (Received for publication April 4, 1942) Following the demonstration by Tocantins and his

Cell Theory Cell Structure and Function Chapter 6 Pg. 94-124 What is a cell? The basic functional unit of all living things. The Cell Theory states All organisms are made of one or more cells. Cells are

The Cell Cell theory (1838): 1. All organisms are composed of one or more cells, and the life processes of metabolism and heredity occur within these cells. 2. Cells are the smallest living things, the

Environmental Health Perspectives Vol. 77, pp. 5-9, 1988 The Use of Rabbits in Male Reproductive Toxicology by Daniel Morton* The rabbit is the smallest and least expensive laboratory animal in which serial

7-2 Eukaryotic Cell Structure & Function These are micrographs of cells you will see in this week's lab. One is plant (onion epithelium), the other animal (human cheek epithelium). Determine which is which

Cell Organelles and their s Chapter 4 Section 2 1. Cell Membrane A phospholipid bilayer around the cell that contains proteins and other markers Separates the cell from its environment Secretes materials

Toxicology An Introduction John Duffus The Edinburgh Centre for Toxicology General considerations Toxicology is the science which lets us know how substances can harm life by physico-chemical reactions

Exam Name MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. 1) All of the following are synthesized along various sites of the endoplasmic reticulum

Organelles copyright cmassengale 1 Organelles Very small (Microscopic) Perform various functions for a cell Found in the cytoplasm May or may not be membrane-bound 2 Animal Cell Organelles Nucleolus Nucleus

Cellular Form and Function Concepts of cellular structure Cell surface Membrane transport Cytoplasm Modern Cell Theory All living organisms are composed of cells. the simplest structural and functional

Cell Injury MECHANISMS OF CELL INJURY The cellular response to injurious stimuli depends on the following factors: Type of injury, Its duration, and Its severity. Thus, low doses of toxins or a brief duration

3 RESULTS As outlined under External contributions (see appendix 7.1), the group of Prof. Gröne at the DKFZ in Heidelberg (Dept. of Cellular and Molecular pathology) contributed to this work by performing

Unit 2:The Cell Section 3: Organelle Structure and Function Mrs. McNamara Biology Organelle-cell part that performs a specific function for the cell Most are surrounded by a membrane Each helps to maintain

CHAPTER 3 SECTION 2 Cells: The Basic Units of Life Eukaryotic Cells BEFORE YOU READ After you read this section, you should be able to answer these questions: What are the parts of a eukaryotic cell? What

Cell Makeup Chapter 4 The Cell: The Fundamental Unit of Life We previously talked about the cell membrane The cytoplasm is everything inside the membrane, except the nucleus Includes Cytosol = liquid portion

TOXICOPATHOLOGICAL AND FUNCTIONAL CHARACTERIZATION OF THYROID GLAND OF RAT IN FLUOROSIS *Shashi A and Parveen Kumar Department of Zoology and Environmental Sciences, Punjabi University, Patiala-147002,

Chapter 10 Summary Nephrolithiasis remains a public health problem around the world, affecting 12% of the adult population. The prevalence and incidence of kidney stones are increasing with global warming,

The most valuable lipid ever? Spermaceti whale oil Used by sperm whales in a special organ in the huge head cavity Largely comprised of cetyl palmitate ww.thisrecording.com www.greenpeace.org Peer Instruction

A Must Read if You Have Children Would you allow your family to brush their teeth with engine degreaser or rat poison? The fact is, they probably are and you don t even know it. Dental hygiene products

Cells Cells are the structural units of all living organisms ranging from unicellular to multicellular organisms. Biochemical activities of cells are dictated by cell shape and specific subcellular structures.

ORGANS OF THE DIGESTIVE SYSTEM OBJECTIVES: 1. List and describe the major activities of the digestive system. 2. Identify and give the functions of the organs in and along the digestive tract. MAJOR ACTIVITIES